SUMMARY Therapeutic advances in vascular calcification may have far-reaching public benefits. Vascular calcification is a frequent complication of diabetes mellitus associated with the increase of morbidity and mortality. Although the precise mechanism has not been determined, vascular calcification is known to be an active process involving ectopic bone formation, in which osteogenic differentiation occurs in the cells transdifferentiated from other lineages. Previous studies have shown that vascular endothelial cells switch cell fate to differentiate into osteoblastic-like cells in vascular calcification. However, it is unknown if reversing this switch by inducing osteoblastic-endothelial transdifferentiation ameliorates vascular calcification. Advanced investigations have shown that the small molecules are able to reprogram and modulate cell fates, and also shown that endothelial-like cells can be transdifferentiated from other lineages. In present proposal, we take advantage of a small molecule identified by using high throughput technology, aiming to induce osteoblastic-endothelial transdifferentiation and investigate the effects on vascular calcification in diabetes mellitus. In preliminary data, we use a high throughput model to identify that GSK3 inhibitor SB216763 converts osteoblasts into endothelial-like cells. We show that SB216763 or limiting GSK3 modulates protein levels of SMAD1 and - catenin and their transcriptional activity to switch the osteoblastic fate for endothelial differentiation. Furthermore, SB216763 treatment reduces EC-origin osteogenic differentiation and decreases calcification in aorta of matrix Gla protein null mouse, an established model of vascular calcification. The treatment of SB216763 also decreases arterial calcification in diabetic Ins2Akita/+ mice without affecting other tissues. Therefore, we hypothesize that GSK3 inhibition induces osteoblastic-endothelial transdifferentiation to ameliorate vascular calcification in diabetes mellitus. In specific Aim 1, we will elucidate the mechanism underlying osteoblastic-endothelial transdifferentiation induced by GSK3 inhibition. In specific Aim 2, we will determine if GSK3 inhibition ameliorates vascular calcification in diabetic mouse model. If successful, it will build osteoblastic-endothelial transdifferentiation as a new concept, and GSK3 inhibitor SB216763 may emerge as a new therapeutic approach to treat calcification in acquired vascular diseases.